Novel 3-phenylquinazolin-2,4(1H,3H)-diones as dual VEGFR-2/c-Met-TK inhibitors: design, synthesis, and biological evaluation

Multitarget anticancer drugs are more superior than single target drugs regarding patient compliance, drug adverse effects, drug-drug interactions, drug resistance as well as pharmaceutical industry economics. Dysregulation of both VEGFR-2 and c-Met tyrosine kinases (TKs) could result in development and progression of different human cancers. Herein, we reported a novel series of 3-phenylquinazolin-2,4(1H,3H)-diones with thiourea moiety as dual VEGFR-2/c-Met TKs. Compared to sorafenib, cabozantinib went behind VEGFR-2 inhibition to target c-Met TK. The dual VEGFR-2/c-Met inhibitory activity of cabozantinib is due to a longer HB domain than that of sorafenib. Based on pharmacophore of cabozantinib analogues, we designed new dual VEGFR-2/c-Met TKs. We synthesized the target compounds via a new single pot three-component reaction. The cytotoxic activity of synthesized compounds was conducted against HCT-116 colorectal cancer cell line. Compounds 3c and 3e exhibited the highest cytotoxic activity against HCT-116 cell line (IC50 1.184 and 3.403 µM, respectively). The in vitro enzyme inhibitory activity was carried out against both VEGFR-2 and c-Met TKs. Compound 3e has the highest inhibitory activity against both VEGFR-2/c-Met (IC50 = 83 and 48 nM, respectively). Docking studies showed that α-oxo moiety in quinazoline ring formed hydrogen bond HB with Met1160 residue in the adenine region of c-Met TK.

Consequently, cabozantinib was selected as a lead compound, building up the pharmacophore for dual VEGFR-2/c-Met inhibition through incorporation of N-acylthiourea moiety as HB domain that mimics dicarboxamide structure of cabozantinib Fig. 5 3 .Quinazolin-2,4(1H,3H)-dione ring was chosen to occupy adenine pocket of the TKs.α-Oxo moiety was involved in several reported TKIs like dovitinib, nintedanib, orantinib, and sunitinib 36 .Therefore, α-oxo moiety of target compounds was designed to augment HB and increase the affinity for both the VEGFR-2 and c-Met TKs.Finally, we different phenyl and heteroaryl rings with variable substituents were designed to occupy allosteric pocket.

Chemistry
The synthetic pathway of 3-phenylquinazolin-2,4(1H,3H)-dione derivatives 2a,b, 3a-h and 4a,b is depicted in Scheme 1 37,38 .The first step involves the synthesis of intermediate benzoyl isothiocyanate by the reaction of 4-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-benzoyl chloride 1 and ammonium thiocyanate under reflux.In the second step, the latter intermediate got readily transformed to the final products by nucleophilic attack of the amino groups of various used reagents such as urea, thiourea, aromatic and hetero amines via a single pot three-component reaction.
The benzoyl isothiocyanate has been employed in synthesis of some new six membered heterocyclic skeletons like thioxo-triazin-2-one and triazine-2,4-dithione, by nucleophilic addition with urea and thiourea, respectively.Herein, an equimolar reaction of benzoyl isothiocyanate with urea and/or thiourea under reflux for 6-8 h, yields an intermediate which underwent cyclodehydration to furnish compounds 2a,b, respectively.The success of cyclization by water elimination was supported by spectral data of the resulting triazines 2a,b.Their IR spectra showed absorption bands at (3406, 3226), (1716, 1655) and 1237 cm −1 for NH's, C=O's, and C=S, respectively.The 1 H-NMR spectrum of triazine 2a exhibited their presence of two tautomeric forms from the downfield signals at 7.98, and 8.10 ppm for the NH protons.In the same manner, 1 H-NMR of triazine 2b represented 8.06 and 8.08 for NH protons.
The formation of the afforded N-benzoyl-N′-phenylthiourea derivatives 3a-h was carried out by the nucleophilic reaction of different aromatic amines with benzoyl isothiocyanate in dioxane and few drops of TEA for 6-10 h.Their chemical structures were supported by IR and 1 H-NMR spectra.For example, the IR bands of compound 3a illustrated the presence of NH, C=O's and C=S at 3243, 1718, 1663 and 1268, respectively.In Similarly, the intermediate benzoyl isothiocyanate reacts with some heterocyclic amines namely 5-amino-1,3,4-thiadiazole-2-thiol and 2-aminobenzothiazole under reflux for 10-12 h to furnish thiadiazole and benzothiazole 4a,b, respectively.

Biology
In vitro antiproliferative activity against HCT-116 HCT-116 colorectal cancer cell line is characterized by overexpression of both VEGFR-2 and c-Met TKs.Consequently, we selected it to study the cytotoxic activity of the target derivatives 9 .We tested the effect of several concentrations of the designed derivatives on HCT-116 cells by using MTT assay.Table 1 represents the IC 50 of all synthesized derivatives as well as that of the reference drug, cabozantinib.

In vitro toxicity against normal cells
The cytotoxic activity of selected two compounds namely, 3c and 3e against WI38 normal cell line was evaluated to study the safety of the designed compounds to the normal cell (Table 2).Although, both tested compounds exhibited higher cytotoxicity against normal cells than cabozantinib, they are better in terms of selectivity index.Compound 3c showed 20 times selective cytotoxicity against the colorectal cancer cells over the normal cells.Moreover, compound 3e showed more than 3 times selective cytotoxicity against the colorectal cancer cells over the normal cells.Compounds 3c and 3e as well as cabozantinib exhibited considerable safety as the values of SI of all of them are more than 2 39 .

In vitro activity against c-Met and VEGFR-2 tyrosine kinases
Based on our rationale design of dual VEGFR-2/c-Met inhibition, we selected the most active cytotoxic derivatives to the HCT-116 cancer cells for scrutinization of their inhibition activity against both VEGFR-2 and c-Met TKs.Table 3 represents the inhibition activity of tested derivatives as well as cabozantinib.All tested derivatives showed acceptable inhibitory activity against the target TKs.Compound 3d exhibited comparable activity to cabozantinib against VEGFR-2 enzyme with (IC 50 = 51 nM).In addition, compound 3e showed noticeable

Apoptosis assay
To go deeper in the mechanism of designed derivatives, the cell cycle arrest study of compounds 3c and 3e was conducted by using Annexin V-FITC/PI staining (Table 4 and Fig. 6).HCT-116 cells were treated by cabozantinib and the two tested compounds at their IC50 concentrations.Compared with the control group, HCT-116 cell cycle was blocked in G0/G1 phase after treatment with 3c (55.41%).On another hand, compound 3e (26.51%) had a higher ability to enhance the population of HCT-116 cells in G2/M process than cabozantinib (24.72%).Moreover, we studied the apoptosis mechanism of HCT-116 that was induced by tested derivatives 3c and 3e as well as cabozantinib (Table 5 and Fig. 7).There was an increase in the number of both early and late apoptotic cells of both tested compounds 3c and 3e.The number of late apoptotic cells of 3e (35.39%) was higher than that of reference drug, cabozantinib (32.13%).Also, the number of late apoptotic cells of 3c (19.28%) was approximately equal to that of cabozantinib (19.61%).Totally, both designed compounds 3c and 3e were able to induce remarkable apoptosis.Moreover, compound 3e succeeded in apoptosis induction more efficiently than cabozantinib (Fig. 8).

In silico studies
Molecular modeling studies Molecular docking of the target compounds was carried out in the active site of c-VEGFR-2 (PDB: 4ASD) and c-Met (PDB: 3LQ8) TKs.Docking styles of compounds 3c and 3e in the active site of VEGFR-2 TK are shown in Figs. 9 and 10, respectively.The thiourea group of compound 3c showed HB with a highly conserved residue

In silico prediction of physicochemical and pharmacokinetic properties
To reach advanced clinical phases, drug candidates have to possess an acceptable pharmacokinetic profile.Therefore, the physicochemical and pharmacokinetic properties of target derivatives were predicted using SwissADME (Tables 6, 7 and Fig. 13).All target compounds showed good druglikeness with no Lipinski violations except compound 3h.Regarding Abbott bioavailability score, most target derivatives have accepted oral absorption.All target compounds as well as cabozantinib were predicted not to cross BBB.Target compounds were predicted to have little effect on CYP450 enzymes like CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 which minimizes the suspected drug-drug interactions.

Conclusion
In brief, based on the structure of a dual VEGFR-2/c-Met inhibitor, cabozantinib, we designed and synthesized a new series of novel 3-phenylquinazolin-2,4(1H,3H)-diones with (thio)urea scaffold.The cytotoxic activity of the target compounds was conducted against HCT-116 colorectal cancer cell line.Compounds contain electronwithdrawing groups on phenyl ring at position-4, declared the highest cytotoxic activity.The inhibitory activity was performed against both VEGFR-2 and c-Met TKs.α-Oxo moiety in quinazoline ring form HB with Met1160 residue in the adenine region of c-Met TK.Compound 3e has the highest inhibitory activity against both VEGFR-2/c-Met (IC 50 = 83 and 48 nM, respectively).

Experimental Chemistry
All reactions were observed on silica gel GF254 plate with thin layer chromatography (TLC).Melting points (uncorrected) were recorded on an electrothermal melting apparatus.FT-IR spectra were recorded on a Shimadzu 8101 PC spectrometer. 1 H-and 13 C-NMR spectra were determined on a Varian Mercury 400 MHz spectrophotometer in DMSO-d 6 .Chemical shifts are reported in parts per million with tetramethylsilane as an internal standard and are given in δ units.Electron impact mass spectra were obtained at 70 eV using a GCMS-QP 1000 EX spectrometer.Elemental analyses were carried out at the microanalytical center at Cairo University.

Figure 6 .
Figure 6.Flow cytometric analysis of cell cycle phases after treatment with 3c, 3e and cabozantinib.
Asp1046 in the HB region and α-oxo group of quinazoline formed HB with backbone amide of Phe918 in the hinge region.Moreover, compound 3c exhibited hydrophobic interactions with both Leu840 and HB with Lys868.4-Oxo moiety of compound 3e declared HB with the conserved residue Cys919 in the hinge region.Compound 3e showed another two HB with Asp1046 and Lys868 in addition to hydrophobic interaction with Leu840 27,28 .On the other hand, Figs.11 and 12 represent docking representations of compounds 3c and 3e it the active site of c-Met TK. α-Oxo moiety of quinazoline ring of compound 3c showed HB with highly conserved residue Met1160 in the hinge region as well as thiourea group formed HB with the another highly conserved residue Lys1110 in the HB region.Further, compound 3c declared hydrophobic interaction with Ile1084.N-acylthiourea group of compound 3e exhibited dual HB with both Lys 1110 and Asp1222 in addition to hydrophobic interactions with both Ile1084 and Met1211 in the hinge region.

Table 1 .
In vitro cytotoxicity of compounds 2a

Table 2 .
In vitro cytotoxicity of compounds 3c and 3e against WI38 normal cell line.

Table 4 .
Cell cycle analysis in HCT-116 colon cancer cell line treated with compounds 3c and 3e.

Table 7 .
Solubility and pharmacokinetics of target compounds and cabozantinib.